Liquid analysis cartridge

ABSTRACT

The present invention provides an apparatus and method for storing a particle-containing liquid. The storage apparatus comprises a microfluidic convoluted flow channel having a plurality of particle capture regions. The storage channel is preferably an isotropic spatially periodic channel. Sedimented particles can be resuspended following storage. This invention further provides a microfluidic analysis cartridge having a convoluted storage channel therein. The sample analysis can use optical, electrical, pressure sensitive, or flow sensitive detection. A plurality of analysis channels can be included in a single cartridge. The analysis channels can be joined to reagent inlets for diluents, indicators or lysing agents. A mixing channel can be positioned between the reagent inlet and the analysis region to allow mixing and reaction of the reagent. The cartridge can include additional valves and pumps for flow management. The analysis cartridge can be a self-contained disposable cartridge having an integral waste storage container. This invention further provides a sheath flow assembly. The sheath flow assembly includes a sample channel and first and second sheath fluid channels positioned on either side of and converging with the sample channel. The assembly also includes upper and lower sheath fluid chambers positioned above and below and converging with the sample channel. The flow cartridges of this invention can be formed by molding, machining or etching. In a preferred embodiment they are laminated. This invention further provides a method of fabricating a laminated microfluidic flow device. In the method, flow elements are formed in rigid sheets and abutting surfaces of the sheets are bonded together.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 09/080,691 filed May 18, 1998.

FIELD OF THE INVENTION

This invention relates to microfluidic cartridges for analysis of liquidsamples, and in particular to cartridges having a convoluted samplestorage channel and to cartridges having a flow cytometric measuringregion.

BACKGROUND OF THE INVENTION

With the advent of micro-machining technology, microfluidic devices haveproliferated (for example, U.S. Pat. No. 5,637,469 to Wilding et al.,U.S. Pat. No. 4,983,038 to Ohki et al., U.S. Pat. No. 4,963,498 toHillman et al., U.S. Pat. No. 5,250,263 to Manz et al., U.S. Pat. No.5,376,252 to Ekstrom et al., E.P. Patent Publication 0381501B1, andPetersen, E. (1982) Proc. of the IEEE, vol. 70, No. 5, pp. 420-457). Apractical limitation for particle-containing liquids such as blood isthe sedimentation of particles within the device. Following loading theliquid in the device, appreciable particle sedimentation can occurwithin the time required to position the device in a measurementapparatus. For example, if the sample flow is slowed or stopped, bloodcells can measurably settle out of plasma within 20 seconds. Without asample management method and apparatus for sedimentation mitigation,quantitative analysis, especially using more than one analysis methodsequentially, is impractical. Moreover, if samples are first collectedand then transported to a measurement apparatus, as in a clinicalsetting or in field sampling, particle sedimentation can make accurateanalysis impossible.

Microfluidic devices having sample storage reservoirs are known in theart (for example, E.P. Patent Publication 0381501B1). Because ofparticle sedimentation, these devices are useful only for sampleswithout particles. Flow cytometric microfluidic devices are also knownin the art (for example, U.S. Pat. No. 4,983,038 to Ohki et al.). Flowcytometric measurements are specifically applicable toparticle-containing liquids. However, without sedimentation mitigationthe measurements can be performed only immediately following samplecollection.

SUMMARY OF THE INVENTION

The present invention provides an apparatus and method for storing aparticle-containing liquid. The storage apparatus comprises a fluidicconvoluted flow channel having a plurality of particle capture regionstherein. Particle capture regions are bends in the channel that providelocal gravitational minima. When sample flow is arrested (i.e. stoppedor slowed) during operation or storage, each of the particles sedimentsin the nearest particle capture region. Unlike a storage reservoir, theparticles do not aggregate in a single clump. Because the particles arelocally captured in a plurality of regions, it is possible to rapidlyand effectively reconstitute the sample following sedimentation. Thestorage channel is preferably spatially periodic, where the termspatially periodic channel is used herein for a channel having asubstantially constant number of particle capture regions per unitvolume. Spatial periodicity facilitates sample reconstitution. Thestorage channel is more preferably an isotropic spatially periodicchannel, where the term isotropic is used herein for a channel suitablefor storing a particle-containing liquid regardless of channelorientation.

The particles can be resuspended by either a continuous or a reversingflow. For resuspension by continuous flow, the arrested sample flow isre-started and particles rejoin the sample fluid. The leading edge andtrailing edge of the sample storage segments are discarded, but themiddle segment is resuspended to a homogeneous mixture identical to theoriginal sample. For the suspension by a reversing flow, a plurality ofresuspension cycles are employed. Each resuspension cycle includes adispense portion to sweep a volume of the stored sample, and an aspirateportion to sweep the volume in the opposite direction. Flow rates, sweptvolume and number of cycle are tailored to the sample fluid.

This invention further provides a fluidic analysis cartridge having aconvoluted storage channel therein. The cartridge contains a sampleinlet, a convoluted sample storage channel in fluidic connection withthe inlet, an analysis channel, having an analysis region, in fluidicconnection with the storage channel, and a valve interface positionedbetween the storage channel and the analysis region. The inlet includesan inlet shut-off interface to prevent leakage of the stored samplethrough the inlet. The cartridge further includes a resuspension pumpinterface to resuspend a sedimented sample by sweeping the sample fromthe storage channel in a continuous or reversing flow. The convolutedstorage channel enables accurate analysis of particle-containingsamples. The sample analysis region provides for detection by any meansknown in the art, for example optical, electrical, pressure sensitive,or flow sensitive detection. For electrical detection, the cartridge caninclude an electrical interconnect. For optical detection, the cartridgecan include a window positioned over the analysis region. The opticalanalysis can employ optical absorption, fluorescence, luminescence orscattering. Particularly useful are absorption and flow cytometricanalyses.

A plurality of analysis channels can be included in a single cartridge.The analysis channels can be joined to reagent inlets to mix the samplewith reagents such as diluents, indicators and lysing agents. Thereagents can be fed into the cartridge using a pump, for example asyringe pump. The reagent can alternatively be stored in a reservoir inthe cartridge. For microscale channels, having laminar flow, mixing ofthe reagent with the sample is predominantly diffusional mixing. Amixing channel can be positioned between the reagent inlet and theanalysis region to allow mixing and reaction of the reagent with thesample. The cartridge can include additional valves and pumps for flowmanagement. The analysis cartridge can be a self-contained disposablecartridge having an integral waste storage container to seal biologicaland chemical waste. The storage container can include a vent to releasegases during fluid loading. The cartridge can have alignment markingsthereon to facilitate positioning in an analysis instrument.

This invention further provides a disposable fluidic hematologycartridge and a method for using the cartridge. The hematology cartridgehas both an absorption measuring channel and a flow cytometric measuringchannel. The cartridge can include a convoluted storage channel. It canfurther include reagent inlets, mixing channels, a waste storagecontainer, and valves and pumps. The flow cytometric measuring channelpreferably has a means for forcing particles in the sample fluid intosingle file. This can be accomplished with a constricted flow passage.It is preferably accomplished using a sheath flow assembly.

This invention further provides a sheath flow assembly. The sheath flowassembly includes a sample channel and first and second sheath fluidchannels positioned on either side of and converging with the samplechannel. The assembly also includes upper and lower sheath fluidchambers positioned above and below and converging with the samplechannel. The sheath fluid channels provide hydrodynamic focusing in thewidthwise direction, and the sheath fluid chambers provide hydrodynamicfocusing in the depthwise direction. Because the assembly provideshydrodynamic focusing, geometric focusing is not required. It is notnecessary for the sample channel to contract in either the widthwise ordepthwise direction. Contracting channels can also be employed.

A sample analysis instrument for use with a fluidic analysis cartridgeis further provided. The instrument includes a cartridge holder, a flowcytometric measuring apparatus positioned for optical coupling with aflow cytometric measuring region on the cartridge, and a secondmeasuring apparatus positioned to be coupled with a second analysisregion on the cartridge. The cartridge holder can include alignmentmarkings to mate with cartridge alignment markings. It can also includepump mechanisms to couple with pump interfaces on the cartridge andvalve mechanisms to couple with valve interfaces on the cartridge.

The convoluted storage channel provides one means for resuspendingparticles sedimented during sample storage. This invention also providesanalysis cartridges having a storage reservoir and an alternativeresuspension means. The resuspension means can be an ultrasonic vibratoracoustically coupled to the reservoir or a mechanical agitator eitherpositioned within the reservoir or mechanically coupled to thereservoir.

The flow cartridges of this invention can be formed by any of thetechniques known in the art, including molding, machining and etching.They can be made of materials such as metal, silicon, plastics andpolymers. They can be formed from a single sheet, from two sheets, or,in a preferred embodiment, from a plurality of laminated sheets. Thisinvention further provides a method of fabricating a laminated fluidicflow channel. In the method, flow elements are formed in rigid sheetsand abutting surfaces of the sheets are bonded together. The term rigidsheet is used herein for a substantially inelastic sheet. A rigidmaterial still exhibits flexibility when produced in thin sheets. Theflow elements can include fluid channels within the plane of the sheet,vias (holes) to route the fluid to the next layer, analysis regions,pump interfaces and valve interfaces. The flow elements can be formed bymethods including machining, such as die cutting or laser ablating, andmolding. The sheets can be bonded together by the use of an adhesive orby welding. They can alternatively be held together with mechanicalcompression.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1, comprising FIGS. 1A-B, is an analysis cartridge with aconvoluted storage channel in (A) plan view and (B) cross section.

FIG. 2, comprising FIGS. 2A-B, shows convoluted storage channels withparticle sedimentation for (A) an anisotropic storage channel and (B) anisotropic storage channel.

FIG. 3, comprising FIGS. 3A-D, are isotropic spatially periodicchannels.

FIG. 4, comprising FIGS. 4A-B, is a pinch valve (A) unactuated and (B)actuated.

FIG. 5 is a syringe pump interface.

FIG. 6 is a plan view of a sheath flow assembly.

FIG. 7, comprising FIGS. 7A-G, shows the individual sheets which arelaminated together to form the sheath flow assembly of FIG. 6.

FIG. 8 shows a reagent channel joining the sample channel.

FIG. 9 shows a convoluted mixing channel following the junction of areagent channel with the sample channel.

FIG. 10, comprising FIGS. 10A-B, illustrates mixing of aparticle-containing sample with a reagent in (A) an anisotropic mixingchannel and (B) an isotropic mixing channel.

FIG. 11 is a schematic drawing of an analysis cartridge having aconvoluted storage channel and a plurality of mixing and analysischannels.

FIG. 12 is a plan view of an analysis cartridge having a convolutedstorage channel, a plurality of reagent inlets, a convoluted mixingchannel, a plurality of analysis regions, a plurality of valve and pumpinterfaces, and a waste storage channel.

FIG. 13, comprising FIGS. 13A-G, shows the individual sheets which arelaminated together to form the analysis cartridge of FIG. 12.

FIG. 14 is a sample analysis instrument for use with a fluidiccartridge.

DETAILED DESCRIPTION OF THE INVENTION

This invention is further illustrated by the following preferredembodiments. In the drawings, like numbers refer to like features, andthe same number appearing in more than one drawing refers to the samefeature. The members of the flow systems of this invention arefluidically connected. The term “between” refers to the fluidicpositioning, which does not necessarily correspond to the geometricpositioning. The terms “top”, “bottom” and “side” refer to theorientation in the drawings, which is not necessarily the orientation ofthe members in operation.

FIG. 1 shows the flow system contained within the cartridge of thisinvention. The term cartridge is used herein for a fluidic device whichis preferably, but not necessarily, disposable and which can be coupledwith measurement, pumping, electronic, fluidic or other apparatus. Itincludes sample inlet 10, convoluted sample storage channel 20,resuspension pump interface 40, sample analysis region 30 and valveinterface 50. The flow system is preferably a microfluidic flow system.The term microfluidic channel is used herein for fluid elementsdimensioned so that flow therein is substantially laminar. In a laminarflow system turbulence is negligible. To maintain laminar flow in thestorage channel, preferably the width of the channel is less than 2000μm and the depth of the channel is less than 300 μm. To prevent cloggingby particles, the dimension must be greater than the largest particledimension, typically greater than 25 μm.

The sample inlet has an inlet shut-off interface to prevent the loadedsample from leaking out of the cartridge. In the illustrated embodimentthe sample inlet comprises a septum. A hypodermic needle is used toinject the sample through the septum. Upon removal of the needle, theseptum forms a shut-off to keep the sample in the flow system.Alternatively, the sample inlet can be a non-sealing inlet such as acapillary or a channel which mates with a sample conduit. If the inletdoes not have an integral shut-off interface, it can be combined with aseparate valve interface.

The resuspension pump interface is used for reconstituting a sedimentedsample following stop flow or storage. The pump can provide continuousor reversible flow. For continuous flow resuspension, the leading edgeand trailing edge of the sample storage segment must be discarded, butthe sample segment in the middle is resuspended to a homogeneous mixtureidentical to the original sample. Significant operating parameters arethe resuspension flow rate and the resuspension time. Reversible flowresuspension uses a plurality of dispense/aspirate cycles. In thisprotocol, in each cycle the sedimented sample is swept through thechannel in dispense mode and then swept back in aspirate mode. The sweptvolume is typically 1-4 periods of the spatially periodic channel. Theaspirated volume is typically equal to the dispensed volume. Thesignificant operating parameters are the resuspend swept volume, thenumber of resuspension cycles and the resuspension flow rate. For eitherprotocol, the resuspension parameters are specific to the particle ladenfluid under consideration and the geometry of the storage channel.Suitable resuspension flow rates and times can be calculated ordetermined empirically.

To calculate the required flow rate, {dot over (V)}, the channelgeometry and fluid properties are considered. For substantiallyrectangular geometries, the critical flow rate is a function of thewidth W and depth D of the channel and of the effective viscosityμ_(eff) of the particulate suspension according to: $\begin{matrix}{V = \frac{2D^{2}W\quad\tau_{crit}}{3\quad\mu_{eff}}} & {{Equation}\quad 1}\end{matrix}$By extrapolation of the data in Alonso et al. (1989), Biorheology 26,229-246, the critical wall shear stress, τ_(crit), for cell suspensionmaintenance is estimated to be 0.14 Pa. As shown by Eq. 1, for greaterchannel dimensions the critical flow rate is greater. For a channel 50μm×100 μm in cross-section, the critical flow rate is 0.008 μl/s. For a300 μm×1000 μm channel, the critical flow rate is 2.8 μl/s.

The valves and pumps of this invention can be entirely incorporated inthe cartridge, or the cartridge can include only valve and pumpinterfaces, and the remainder of the valve and pump mechanisms can beexternal to the cartridge. A pump (valve) comprises a pump (valve)interface and a pump (valve) mechanism. The interface is that portionwhich is directly connected to flow elements, and the mechanism is theexterior portion. The cartridge can be inserted in measurement apparatuscomprising valve and pump mechanisms. Upon loading the cartridge in theapparatus, the valve and pump mechanisms engage the valve and pumpinterfaces. The valves can be either normally open or normally closed.They can be manually or automatically actuated.

Sedimentation in convoluted storage channels is illustrated in FIG. 2.When the flow is arrested the particles sediment in the nearest particlecapture region, which are bends at gravitational potential minima. Thegravity vector is illustrated in the drawings. The channels contain aplurality of particle capture regions so that the particles cannotaggregate in a single clump. The illustrated convoluted channels arespatially periodic. The term spatially periodic channel is used hereinfor a channel having a substantially constant number of particle captureregions per unit volume. This facilitates recreating a homogeneoussample upon resuspension. The illustrated embodiments are spatiallyperiodic in a conventional geometric sense, having repeating units oflength λ. Alternatively, the channel can be randomly convoluted butnonetheless have a substantially constant number of particle captureregions per unit volume.

The channel of FIG. 2A is suitable for storing particle-containingliquid in the illustrated orientation. If it were aligned along thechannel axis, i.e. rotated so that the inlet and outlet were at the top,all of the particles would accumulate in the bottom capture region andwould be difficult to resuspend uniformly. This type of spatiallyperiodic channel is referred to herein as anisotropic because thesuitability for storage depends on orientation. This anisotrophy can bedisadvantageous. To prevent clumping the cartridge must be carefullyhandled to ensure that it is never aligned along the channel axis.

The channel of FIG. 2B can be used for storage at any orientation and isthus referred to herein as an isotropic storage channel. Isotropicchannels are preferred because it is not necessary to maintain aparticular orientation during handling. Further examples of isotropicspatially periodic channels are shown in FIG. 3. The channel of FIG. 3Ahas the same structure as the channel of FIG. 2B but with more repeatedunits. The channel of FIG. 3B is similar but with rounded corners. Thiscan be advantageous for manufacturing and assembly. The channels ofFIGS. 3C and D are referred to as “omega” channels, angular in FIG. 3Cand rounded in FIG. 3D. Omega channels are similar to the square wavechannel of FIG. 2A except that bringing the bases of the square wavetoward one another adds additional capture regions, and thereby makesthe channel isotropic. FIG. 3 shows a few examples of storage channels;numerous other isotropic spatially periodic channels can be utilized. Inthe following schematic drawings square waves are used as a genericillustration of convoluted channels. Other embodiments may be preferredand in particular isotropic channels may be preferred.

This invention also provides a structure containing an isotropic storagechannel. The structure is any solid material with a channel formedtherein. The structure can be a disposable cartridge or a permanentlyinstalled element of a measurement or reaction instrument. It can be amicroscale channel dimensioned for laminar flow or a macroscale channeldimensioned for turbulent flow. One embodiment is a bioreactor whereinreagents, which can include cells, are incubated in the channel followedby resuspension of particles.

A preferred embodiment of valve interface 50 is shown in FIG. 4. FIG. 4Ashows a cross-section of the valve in the open position and FIG. 4Bshows the valve in the closed position. Channel 21, running orthogonalto the plane of the paper, has walls formed by sheet 162B, and top andbottom formed by sheets 162A and C. Elastic seal 51 fits within anopening in sheet 162A. The fluid element containing sheets aresandwiched between upper cartridge case 130 and lower cartridge case131. The valve mechanism includes valve pin 150 which is made of a rigidmaterial, for example metal or plastic. The valve pin is guided by anopening in upper case 130. When actuated, the pin presses against seal51, which extrudes into the channel, thereby closing it. Note thatalthough it is termed a pinch valve, the channel itself is not pinchedclosed. The valve mechanism can be incorporated into the cartridge or itcan be a separate element. Seal 51 is made of a deformable material suchas silicone, urethane, natural rubber or other elastomers. In theillustrated embodiment, the channel is formed with three separatesheets, 162 A-C; it can instead be formed in fewer than or in more thanthree sheets. The pinch valve of FIG. 4 is an example of a valve thatcan be used with the analysis cartridge. Other valves can instead beused.

An embodiment of resuspension pump interface 40 is shown incross-section in FIG. 5. Channel 22A, running orthogonal to the plane ofthe paper, has walls formed within sheet 164B and bottom formed by sheet164C. Fluid communication via 22 is a circular hole in sheet 164Aallowing fluid flow from 140 to 22A. Elastic seal 41 fits between sheet164A and upper cartridge case 130. The pump mechanism includes cannula140, which is preferably connected to a syringe pump, not shown. Thecannula can be inserted into seal 41 to introduce fluids into channel22A. The cannula can be essentially a needle with a polished tip toavoid damaging the seal. In the resuspension procedure, a fluid such assaline or water is it injected into the channel through the cannula, andit sweeps the sample fluid through the channel. To reverse the flow, thesaline in extracted through the cannula. The syringe pump interface canbe used both as a pump, one- or two-directional, and as a reagent inlet.The entire pump, interface and mechanism, can be incorporated in thecartridge, or only the interface can be incorporated and the mechanismcan be separate.

The sample analysis region provides for detection by any means known inthe art, for example optical, electrical, pressure sensitive, or flowsensitive detection. More then one analysis means can be employed in asingle analysis region, for example optical and electrical. Forelectrical detection, the cartridge can include an electricalinterconnect. The cartridge can be electrically connected to electricalmeasuring apparatus. For optical detection, the cartridge can include awindow positioned over the analysis region for optical coupling withmeasuring apparatus such as light sources and photodetectors. Thewindows can be inserted glass or, if the channel is formed intransparent sheets, the sheets themselves can serve as windows. Theoptical detection can be absorption, luminescent, fluorescent orscattering based. The cartridge can comprise a plurality of sampleanalysis regions. One of the analysis regions can provide a fillingstatus gauge to indicate that the storage channel is filled. The gaugecan be based on optical absorption measurement, pressure measurement,conductivity measurement, flow measurement or any measurement thatindicates the presence of a fluid in the gauge. For absorptionmeasurement, visual observation of filling status may be used.

In a preferred embodiment, the analysis region is a flow cytometricanalysis region. Preferably a sheath flow assembly is positioned alongthe analysis channel before the flow cytometric analysis region. FIGS. 6and 7 illustrated a preferred embodiment of the sheath flow assembly.The assembly comprises seven sheets, 166A-G, which are laminatedtogether to form the fluidic elements of analysis cartridge 160. Theanalysis channel, comprising core stream channel 26 and sheathed streamchannel 27, is connected to the convoluted storage channel (not shown).In sheath flow assembly 70, first and second sheath fluid channels,jointly labeled as element 72 (FIG. 7D), are positioned on either sideof and converge with channel 26. In this embodiment the diameter of thesheathed portion is greater than the core portion of the analysischannel. The sheath fluid channels extend into layers 166C and E, andare labeled as elements 75 and 76. The sheath fluid channels providehydrodynamic focusing of particles in channel 27 in the widthwisedirection. Upper and lower sheath fluid chambers 73 and 74 are formed insheets 166B and F. When assembled, they are positioned above and belowand converge with channel 26. The sheath fluid chambers providehydrodynamic focusing in the depthwise direction. To minimize layer tolayer depthwise discontinuities in the region where the sheath fluidchannels and chambers converge with the analysis channel, the downstreamedges are staggered. The edge of channels 75 and 76 are slightly to theright of the edge of channel 72. Sheath fluid is conducted to the sheathflow assembly through sheath fluid channel 71 (FIG. 7B). Vias 77 insheets 166C-E connect channel 71 with the sheath fluid chambers Thesheath fluid chambers communicate fluid to the sheath fluid channels. Intypical hydrodynamic focusing operation, the ratio of sheath flow tocore stream 26 flow is around 130:1.

Following hydrodynamic focusing, flow cytometric measuring is performedin analysis region 30. The analysis region includes window recesses 31and 32 in sheets 166C and E positioned above and below the focusedsample. The window recesses accommodate glass inserts. In lieu ofrecesses, sheets 166C and E can themselves serve as windows. In theremaining sheets, optical clearing holes 33 allow optical access to theanalysis region. The sheets in FIG. 7 are sandwiched between an uppercase and a lower case. Layers 166A and G can be incorporated in thecase. The illustrated embodiment also includes waste storage container100. It is connected with flow channel 27 through vias 101 and to a casemounted storage container through vias 102.

One embodiment of the sheath flow assembly has been illustrated. Othersheath flow assemblies known in the art can be utilized, for exampleU.S. Pat. No. 4,983,038. Because this sheath flow assembly of thepresent invention provides both widthwise and depthwise hydrodynamicfocusing, geometric focusing is not required. Although not necessary,the analysis channel can decrease in width and/or depth and in adownstream direction. Two-dimensional hydrodynamic focusing can also beachieved using the device of U.S. patent application Ser. No.08/823,747, filed Mar. 26, 1997. In lieu of hydrodynamic focusing theflow channel can be constricted in the analysis region to provide singlefile particles, as described in single file, as described in U.S. Pat.No. 5,726,751.

Another preferred embodiment of the sample analysis region is anabsorption analysis region. For increased sensitivity using anabsorbance based assay the optical pathlength, i.e. the channel depth,in the absorption measurement region is increased. For decreasedsensitivity to factors such as intermittent sample stream perturbations,optical window quality and optical measurement apparatus lens defects,the effective illumination area of the detection region can be increasedby increasing the channel width. There is a design trade-off betweenincreasing the channel width and depth and minimizing the volume of themicrofluidic system. This balance can be determined for a specificassay, a specific set of light sources, detectors and optics, and therequired accuracy and resolution.

The cartridge can also include an inlet for mixing a reagent with thesample fluid prior to sample analysis, as shown in FIG. 8. The term“reagent” refers to any fluid that joins the sample fluid. It can be,for example, a diluent, a lysing agent, an indicator dye, a fluorescentcompound, a fluorescent standard bead for flow cytometric calibration,or a reporter bead for flow cytometric measurement (U.S. Pat. No.5,747,349). Between storage channel 20 and analysis region 30, reagentchannel 80 joins analysis channel 24. The reagent channel is connectedto pump interface 40A and reagent inlet 60. In a preferred embodimentthe pump and the inlet are combined in a syringe pump. The cartridgeincludes valve interface 50 to separate the storage channel from thereagent inlet.

When the flow channels are microchannels having laminar flow therein,mixing between the reagent and the sample is predominantly diffusionalmixing. The streams can join in side-by-side flow, as described in U.S.Pat. No. 5,716,852 and U.S. Ser. No. 08/829,679 filed Mar. 31, 1997, orin a layered flow for more rapid mixing, as described in U.S. Pat. No.5,972,718 issued Oct. 26, 1999, and U.S. Ser. No. 08/938,585 filed Sep.26, 1997. In order to allow for mixing and reaction prior to analysis, amixing channel can be included, as shown in FIG. 9. Mixing channel 90 ispositioned between the reagent inlet and the analysis region. Thegeometry of mixing channel 90 is selected to allow mixing and reactionbetween the sample and reagent streams. The mixing channel can beconvoluted in order to achieve the desired time delay within a compactspace. Alternatively, active mixing methods can be employed, includingultrasonic, mechanical, sonic, flow induced, etc.

In the embodiment of FIG. 9 the mixing channel is illustrated as asquare wave. For a particle-containing sample, it may be desired toallow diffusional mixing between smaller species within the sample andreagent streams without allowing particles in the sample screen togravitationally settle into the reagent stream. FIG. 10 shows the effectof channel geometry on gravitational mixing. A square wave channel isillustrated in FIG. 10A. The particle-containing sample stream entersmixing channel 90 through channel 24 and reagent stream enters throughchannel 80. In the upper half of the mixing channel the sample stream isgravitationally above the reagent stream and particles tend to settleinto the reagent stream. In the lower half of the mixing channel this isreversed and particles settle back into the sample stream. This reversalof top and bottom for the sample stream and reagent stream can be usedmore effectively in an isotropic channel as illustrated in FIG. 10B. Ina spatially periodic isotropic channel the gravitational top and bottomof the channel interchange within each repeating unit. This counteractsthe effect of gravity on the particles in the sample stream. Theisotropic spatially periodic channel is therefore useful forsedimentation mitigation as well as sedimentation resuspension.

The cartridge can provide for more than one analysis region, in seriesor in parallel. Multiple parallel analysis regions are illustratedschematically in FIG. 11. The device of FIG. 11 comprises sample inlet10, storage channel 20, resuspension pump interface Pl1 (Pump Interface1), and analysis regions 30A-C. At junctions J1, J3, J5, J6 and at theend of the storage channel, fluid from the sample storage channel can bedirected to analysis channels 24A-D and to waste storage container 100.Note that in this embodiment the resuspension pump is fluidicallyconnected to the storage channel in the middle of the channel ratherthan at the beginning of the channel 1. Preferably the sample segmentbetween J1 and J3 flows through valve V3 for analysis, the samplesegment between J3 and J5 flows through valve V2 for analysis and thesegment between J5 and J6 flows through valve V1 for analysis.

The cartridge further includes pump interfaces PI2-PI5, valve interfacesV1-V5, reagent channels 80A-C, sheath flow assembly 70, waste storagecontainer 100, and vents 110A-C. In a preferred embodiment, the sampleinlet is a septum, the pump interfaces are syringe pump interfaces andthe valve interfaces are pinch valve interfaces. The vents are made ofgas permeable plugs having a reduced permeability when wet. The storageand mixing channels are illustrated as square waves but are preferablyisotropic spatially periodic channels. The sheath flow assembly ispreferably as illustrated in FIGS. 6 and 7. Analysis region 30C is afilling status gauge providing visual indication of proper sample load.Analysis region 30A is an absorption measurement region, opticallycoupled with measurement apparatus comprising both a green and a blueLED and a photodetector. Analysis region 30B is a flow cytometricanalysis region optically coupled with a measurement apparatuscomprising a diode laser and a plurality of photodetectors at variousoptical axis and collection cone angles.

The cartridge of FIG. 11 can be used for hematology. A single cartridgecan determine the red cell count, the total hemoglobin, and the whitecell count and characterization. The analysis requires only 15 μl ofsample, and the waste fluid is contained within the cartridge for safeoperation and disposability. The sample is loaded into the storagechannel through inlet 10. At J1 the potentially contaminated leadingedge of the sample flows in bypass channel 25 (FIG. 12), having a largerdiameter than channel 20. Air in the channel escapes through vent 110A.The next segment of the sample fills the storage channel. Valve V4 isopen and the sample flows to filling status indicator 30C. Vent 110Callows air to escape during sample loading. Excess sample flows intosample load bypass storage 115. The cartridge can be stored ortransported prior to analysis. For measurement the cartridge is insertedinto a measurement instrument having a cartridge holder and valve andpump mechanisms, which engage the valve and pump interfaces on thecartridge. The pump mechanisms comprise syringe pumps wherein thesyringes are filled with reagents. The syringe connected to PI1 isfilled with an inert driving fluid, the syringe connected to PI2 isfilled with diluent, the syringe connected to PI3 is filled with a softlysing agent, the syringe connected to PI4 is filled with a Drabkinlysing reagent and the syringe connected to PI5 is filled with a sheathfluid.

After insertion in the measurement apparatus, the sample is resuspendedand analyzed. The entire measurement, including sample resuspension, canbe performed in less than two minutes. The -procedure-for operating theanalysis cartridge of FIG. 11 for hematology-is outlined in Tables 1-3.For each time interval from t1 through t17, Table 1 describes theprocedure, Table 2 gives the elapsed time, and Table 3 gives the statusof valves and pumps fluidically connected to the cartridge and thestatus of optical measurement apparatus optically connected to thecartridge. In the first analysis time interval, t1, air is purged fromresuspension pump interface PI1 through valve V5 into waste storagecontainer 100. In t2 the reagent and sheath fluid channels are purgedand wet. In t3 the optical path in absorption measurement region 30A iscalibrated using the blue LED. In t4 the total hemoglobin sample segmentbetween J1 and J3 is resuspended by alternating dispense and aspiratecycles using P1. In t5 the total hemoglobin assay is performed by mixingthe blood with Drabkin reagent to lyse the red blood cells, andmeasuring the absorption in analysis region 30A. To create a bubble-freemixture in the analysis region, air is purged from channels 24A and 80A.Preferably the sample fluid and the reagent reach J2 simultaneously.Mixing channel 90A is designed to allow formation of thecyanomethahernoglobin complex.

Following hemoglobin absorption assay, flow cytometric analysis isperformed. In time intervals t6, t7 and t8 the channels used in flowcytometric analysis are purged. To protect optical surfaces in thecytometric region from direct contact with the sample, sheath fluid ispumped through the region during the purge. The sheath flow is set to alow ratio to minimize fluid accumulation in the waste storage containerduring priming stages. In t9 the RBC sample segment between J5 and J6 isresuspended. In t10 and t11 the optical measuring apparatus is alignedand the flow is stabilized. In t12 and t13 the RBC flow cytometric assayis performed. In t14 the WBC sample segment between J3 and J5 isresuspended. In t15 a soft lysing reagent is added to the sample andtime is allowed for mixing and reaction in mixing channel 90B. In t16and t17 the WBC assay is performed. The total elapsed time is 1.75minutes. Following analysis, the cartridge is disposed of.

Drawings of a preferred embodiment of the hematology cartridge are shownin FIGS. 12 and 13. FIGS. 13A-G show the seven sheets, 167A-G, which arelaminated together to form cartridge 160 shown in FIG. 12. This is athree-dimensional fluidic structure wherein channels in different layersappear to overlap in FIG. 12 but are in fact separated by sheets 167Cand E. Vias in intervening sheets connect flow elements in differentlayers. Three-dimensional structures can be more compact and rugged thantwo-dimensional structures. Registry of the laminated sheets to the caseis accomplished with holes 170 in the sheets. The case has pins that fitwithin holes 170. For measurement, the cartridge is inserted into ameasurement instrument including a cartridge holder. The outer case ofthe cartridge (not shown) has alignment markings thereon for optical andfluidic alignment with the measurement apparatus. In this embodiment,the alignment markings are kinematic alignment markings comprising apit, a groove and a flat. The cartridge holder has corresponding pins.The shape of the cartridge is designed for engagement with the cartridgeholder, and thus in itself comprises an alignment marking.

Sample is introduced through inlet 10 and stored in channel 20. Thesample leading edge flows into bypass channel 25. The bypass channel isfluidically connected to a case-mounted waste storage container (notshown). Syringe pump interfaces 40A-E and pinch valve interfaces 50A-D(FIG. 13A) control sample management in the cartridge. The syringe pumpinterfaces are also reagent inlets. When valve 50D is open sample flowsthrough channel 24D (FIG. 13F) to filling status gauge 30C. For totalhemoglobin assay lysing reagent is introduced through syringe pumpinterface 40D and the mixture flows through analysis channel 24A (FIG.13D) to absorption analysis region 30A. For RBC assay, valve 50A isopened, diluent is introduced through syringe pump interface 40B, andthe red blood cells are hydrodynamically focused in sheath flow assembly70 and counted in flow cytometric analysis region 30B. For WBC assay,valve 50B is opened, a soft lysing agent, which masks red blood cellsand platelets, is introduced through syringe pump interface 40C, mixingand reaction occur in mixing channel 90 (FIG. 13B), the sample ishydrodynamically focused in sheath flow assembly 70 and analyzed in flowcytometric analysis region 30B. Waste fluid from all three analysisregions flows into waste storage container 100 (FIG. 13F), which isfluidically connected with a case-mounted storage container having avent therein. This waste storage container is a channel. It canalternatively or in addition be a fixed or expandable reservoir.

In this embodiment, storage channel 20 and mixing channel 90 are formedin sheet 167D. After cutting the sheet to form the channels, peninsulasof sheet material remain around the channels. The peninsulas are notwell supported and can flop around during laminate assembly. A lessfloppy channel can be formed using two or more layers, with alternatingloops of the channel formed in different layers.

The cartridge has been illustrated with particular mixing andmeasurement configurations. It can also provide filtering, diffusionbased filtering as described in U.S. Pat. No. 5,932,100 issued Aug. 3,1999, simultaneous particle separation and chemical reaction asdescribed in U.S. Ser. No. 08/938,585 filed Sep. 26, 1997, valvelessmicroswitching as described in U.S. Pat. No. 5,726,404, diffusion-basedchemical sensing as described in U.S. Pat. No. 5,716,852, U.S. Pat. No.5,948,684 issued Sep. 7, 1999, and adsorption-enhanced differentialextraction as described in U.S. Pat. No. 5,971,158 issued Oct. 26, 1999.The channel can also include fluidic elements for extraction,electrophoresis, electrochemical reactions, chromatography and ionexchange reactions.

The cartridge can be fabricated from any moldable, machinable oretchable material. The term machining as used herein includes printing,stamping, cutting and laser ablating. The cartridge can be formed in asingle sheet, in a pair of sheets sandwiched together, or in a pluralityof sheets laminated together. The term “sheet” refers to any solidsubstrate, flexible or otherwise. The channels can be etched in asilicon substrate and covered with a cover sheet, which can be atransparent cover sheet. In a laminated embodiment, the channel wallsare defined by removing material from a first sheet and the channel topand bottom are defined by laminating second and third sheets on eitherside of the first sheet. Any of the layers can contain fluid channels.In some cases the channel is simply a hole (or fluid via) to route thefluid to the next fluid laminate layer. Any two adjacent laminate layersmay be permanently bonded together to form a more complex single part.Often fluidic elements that have been illustrated in two separate layerscan be formed in a single layer.

Each layer of a laminate assembly can be formed of a different material.The layers are preferably fabricated from substantially rigid materials.A substantially rigid material is inelastic, preferably having a modulusof elasticity less than 1,000,000 psi, and more preferably less than600,000 psi. Substantially rigid materials can still exhibit dramaticflexibility when produced in thin films. Examples of substantially rigidplastics include cellulose acetate, polycarbonate, methylmethacrylateand polyester. Metals and metal alloys are also substantially rigid.Examples include steels, aluminum, copper, etc. Glasses, silicon andceramics are also substantially rigid.

To create the fluidic element in the sheets, material is removed todefine the desired structure. The sheets can be machine using a laser toablate the material from the channels. The material can be removed bytraditional die cutting methods. For some materials chemical etching canbe used. Alternatively, the negative of the structure desired can bemanufactured as a mold and the structure can be produced by injectionmolding, vacuum thermoforming, pressure-assisted thermoforming orcoining techniques.

The individual layers, assemblies of layers, or molded equivalents arebonded together using adhesives or welding. Alternatively, mechanicalcompression through the use of fasteners such as screws, rivets andsnap-together assembly can be used to seal adjacent layers. Layers canbe assembled using adhesives in the following ways. A rigid contactadhesive (for example, 3M1151) can be used to join adjacent layers. Asolvent release adhesive may be used to chemically bond two adjacentplayers. An ultraviolet curing adhesive (for example, Loctite 3107) canbe used to join adjacent layers when at least one layer is transparentin the ultraviolet. Precision applied epoxies, thermoset adhesives, andthermoplastic adhesives can also be used. Dry coatings that can beactivated to bond using solvents, heat or mechanical compression can beapplied to one or both surfaces. Layers can be welded together. Forwelding the layers preferably have similar glass transition temperaturesand have mutual wetting and solubility characteristics. Layers can bewelded using radio frequency dielectric heating, ultrasonic heating orlocal thermal heating.

The device of FIGS. 12 and 13 was fabricated as follows. Layers 167A andG were made of 4 mil mylar and layers 167C and E were made of 2 milmylar. Layers 167B, D and F were made of 2 mil mylar with 3M1151 on bothsides (4 mil inclusive). The adhesive had cover sheets thereon. With thecover sheets still on the adhesive, the sheets were laser ablated tomachine flow elements therein. The cover sheets were removed and theindividual laminate was assembled with the aid of an alignment jig.

This invention further includes a sample analysis instrument for usewith an analysis cartridge, in particular a hematology analysiscartridge. The instrument has a cartridge holder, a flow cytometricmeasuring apparatus positioned to be coupled with a flow cytometricmeasuring region on the cartridge, and a second measuring apparatuspositioned to be coupled with a second measuring region on thecartridge. The flow cytometric measuring apparatus comprises a lightsource, preferably a laser, and at least one photodetector. Thephotodetectors can be positioned for measuring small angle scattering,large angle scattering or fluorescence. The apparatus can also includeoptical elements such as focusing and collection lenses, wavelengthfilters, dichroic mirrors and polarizers. The second measuring apparatuscan be any measuring apparatus including optical, electrical, pressuresensitive and flow sensitive apparatus. Absorption measuring apparatuscomprising a light source and a photodetector is preferred. Preferablythe light source is positioned on a first side of the cartridge holderand the photodetector is positioned on the opposite side.

A measurement instrument is shown schematically in FIG. 14. It comprisescartridge holder 190, flow cytometric measurement apparatus 180B andabsorption measurement apparatus 180A. Cartridge 160, shown in phantom,slides into the cartridge holder. The measurement apparati arepositioned to be optically coupled with flow cytometric analysis region30B and absorption analysis region 30A. This instrument also includespump and valve mechanism manifold 141. The pump mechanisms are syringepumps which couple to pump interfaces on the cartridge via cannulas 140.The manifold can also include reagent reservoirs to refill the syringepumps for multiple assays. The valve mechanisms activate valve pins 150,which couple to valve interfaces on the cartridge.

Preferably the cartridge holder has alignment markings thereon to matewith corresponding markings on the cartridge. The alignment markings canbe the shape of the holder, protruding pins, notches, rods, kinematicmounts, two stage kinematic mounts as described in U.S. Pat. No.5,748,827 issued May 5, 1998, or any other feature that facilitatespositioning of the cartridge. In lieu of or in addition to cartridgealignment, the instrument can include optical steering elements, such asmirrors, to align the measuring apparatus with the analysis region. Theanalysis instrument can further include valve and pump mechanisms whichcouple with valve and pump interfaces on the cartridge.

All references cited herein are incorporated by reference in theirentirety.

Preferred embodiments described above are intended to be illustrative ofthe spirit of this invention. Numerous variations and applications willbe readily apparent to those skilled in the art. The range and scope ofthis patent is defined by the following claims. TABLE 1 Time IntervalDescription t1 Purge air from PI1 through valve V5. t2 Purge air and wetdelivery lines from PI2 to J7; PI3 to J7; PI4 to J2; and PI5 to J8 t3THB optical path calibration using 430 nm blue LED and Drabkin reagentabsorbtion. t4 THB Sample segment resuspension t5 Total hemaglobinassay; purge of air from J1 to J2 & uniform mixing of sample + Drabkin &creation of a bubble free mixture in flow cell. Time allowed for thecreation of the Cyanomethahemaglobin complex. t6 RBC sample segmentmis/air purge from J6 through J9&J7 to J8. Sheath pump is set to a lowratio, about 5:1 in order to protect optical surfaces of the cytometersection. t7 WBC sample segment mis/air purge from J3 through J4&J7 toJ8. Sheath pump is set to a low ratio, about 5:1 in order to protectoptical surfaces of the cytometer section. t8 J7 junction purge. Purgeair from the region around J7 through the cytometer to waste. t9 RBCsample segment resuspension t10 Beam steering/optical targeting. t11 RBCassay flow stabilization algorithm based on mean pulse frequency PIDfeedback control t12 RBC assay. t13 Second RBC assay (if required) t14WBC sample segment resuspension t15 WBC assay flow stabilization and 15second time delay. t16 WBC assay. t17 Second WBC assay (if required)

TABLE 2 Time interval t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 t13 t14 t15t16 t17 Interval 1 3 2 3 10 2 2 1 3 5 4 4 3 1.6 17 22 22 time(s) Elapsed1 4 6 9 19 21 23 24 27 32 36 40 43 45 62 83 105 time(s) Elapsed 0.020.07 0.10 0.15 0.32 0.35 0.38 0.40 0.45 0.53 0.60 0.67 0.72 0.74 1.031.39 1.75 time (min)

TABLE 3 Resource Status Time interval t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11t12 t13 t14 t15 t16 t17 Resuspension X X X X X X X X X X X X X X pump,P1D (dispense) Resuspension X X X X X X X X X X X X X X pump, P1A(asperiate) Diluent pump, X X X X X X X X X P2 Soft Lyse X X X X X Xpump, P3 THB pump, P4 X X X X Sheath pump, X X X X X X X X X X X X X P5RBC Valve, V1  C¹ O C C C O C O O O O O O O C C C WBC Valve, V2 C O C CC C O O C C C C C C O O O THB Valve, V3 C O C O O C C C C C C C C C C CC Waste C C C C C C C C C C C C C C C C C Isolation Valve, V4 Sample O CC C C C C C C C C C C C C C C delivery purge, V5 Beam Steering X Motor,M1 Beam Steering X Motor, M2 Diode laser X X X X X X X X Green LED XBlue LED X¹C = Closed, O = Open

1-122. (canceled)
 123. A fluidic sample analysis cartridge for analyzinga particle-containing liquid sample, comprising: a sample inlet; amicrofluidic convoluted sample storage channel in fluidic connectionwith the sample inlet, wherein the sample storage channel comprises aplurality of particle capture regions; a first analysis channel influidic connection with the sample storage channel, wherein the firstanalysis channel comprises a first analysis region; and a sheath flowassembly in fluidic connection with the first analysis channel upstreamof the first analysis region.
 124. The cartridge of claim 123 whereinthe sample inlet comprises an inlet shut-off interface.
 125. Thecartridge of claim 123, further comprising: a reagent inlet; and areagent channel in fluidic connection with the reagent inlet and thefirst analysis channel upstream of the first analysis region and thesheath flow assembly.
 126. The cartridge of claim 123 further comprisinga waste storage container in fluidic connection with the first analysischannel.